This proposal aims to develop highly specific, rapid and cost-effective immunoassays to detect Zika virus (ZIKV) infection. ZIKV is a single-stranded RNA virus in the Flaviviridae family that is transmitted to humans through infected mosquitos, blood transfusions, and sexual contact. As of June 2016, around 500,000 Zika Virus disease cases have been estimated in Brazil alone. Recently, ZIKV has been detected in many other countries including the USA and most European countries. The FDA has recently mandated screening the entire US blood supply for ZIKV. Key challenges in ZIKV surveillance include the proportion of cases that remain asymptomatic and the nonspecificity of ZIKV symptoms. Current methods available for detection of ZIKV are not rapid, and furthermore, suffer from problems of unreliability and risks of cross-reactivity. A highly specific, easily performed antibody test is urgently needed for both surveillance and patient care. Mediomics has developed and commercialized a new assay platform (PINCER assays) that allows simple homogenous mix-and-read detection of a variety of targets. These rapid-result assays can reach high specificity while remaining low cost and easy to use. In a recent collaboration with the CDC, Mediomics developed a PINCER-based homogeneous assay prototype for hepatitis C (HCV) that achieved 97.3% specificity, 98.7% sensitivity, and inter and intra assay CV?s better than 10%. These promising analytical parameters, combined with the simplicity of homogenous detection, indicate a great potential for this assay platform. In addition, our long term collaborator, Dr. Tomasz Heyduk?s laboratory, developed a novel screening platform by combining both ribosome display and Next Generation Sequencing (NGS) technologies to quickly identify linear and conformational epitopes of antibodies from patient samples. We hypothesize that by combining our PINCER platform and the peptide epitopes unique to the ZIKV infection, we will be able to develop a specific, rapid, and cost effective immunoassay for ZIKV detection. Toward this goal, we have three specific aims in our Phase I project. Aim 1. Develop a PINCER-based homogeneous immunoassay for ZIKV detection. Aim 2. Explore entire protein sequence space of ZIKV to identify peptide ligands specifically recognized by ZIKV antibodies in patient blood samples. Aim 3. Optimize and evaluate the performance of both peptide and improved antigen-based PINCER assays. A third party will perform the evaluation of the prototype assay. If this project is successful, we should have established a solid foundation for a Phase II project to fully develop and validate a novel immunoassay that is simple (mix-and-read), rapid (10-30 minutes), cost-effective (<5-10% of ELISA), and suitable for low to high throughput screening of ZIKV infection.